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FEBS 29151 FEBS Letters 579 (2005) 455–463

PREL1 provides a link from Ras signalling to the via Ena/VASP proteins

Andrea Jenzoraa, Barbara Behrendtb, J. Victor Smallc,Ju¨rgen Wehlanda, Theresia E.B. Stradala,* a Department of Biology, German Research Centre for Biotechnology (GBF), 38124 Braunschweig, Germany b University Hospital Eppendorf, Institute for Biochemistry and Molecular Biology II (IMB II): Molecular , 20246 Hamburg-Eppendorf, Germany c Institute of Molecular Biotechnology (IMBA), Dr. Bohr-Gasse 3-5, 1030 Vienna, Austria

Received 4 October 2004; revised 18 October 2004; accepted 25 October 2004

Available online 16 December 2004

Edited by Lukas Huber

podia and filopodia harbouring components directly driving Abstract Ena/VASP family proteins are important modulators of and localize to focal adhesions, stress fibres and actin nucleation via the Arp2/3 complex [7,8], and other fac- the very tips of lamellipodia and filopodia. Proline-rich proteins tors exerting modulatory functions [9, reviewed in 10,11,12]. like and zyxin are well established interaction partners, Bar-Sagi and Feramisco showed already in 1986 [13] that which mediate Ena/VASP-recruitment via their EVH1-domains injection of constitutively active Ras into fibroblasts induces to focal adhesions and stress fibres. However, it is still unclear, membrane ruffling, providing a first link between Ras signalling which binding partners Ena/VASP proteins may have at lamelli- and the actin cytoskeleton. The ability of active Ras to increase podia tips and how their recruitment to these cellular protrusions the invasive potential and of fibroblastic cells was pro- is regulated. Here, we report the identification of a novel protein posed to be mediated by the suppression of integrin activation with high similarity to the C. elegans MIG-10 protein, which we and adhesion [14, reviewed in 15]. On the contrary, the Ras termed PREL1 (Proline Rich EVH1 Ligand). PREL1 is a 74 family member Rap1 was found to reverse the phenotype of kDa protein and shares homology with the Grb7-family of sig- nalling adaptors. We show that PREL1 directly binds to Ena/ Ras transformation, to induce adhesion and to trigger integrin VASP proteins and co-localizes with them at lamellipodia tips activation in response to its activation [16, reviewed in 17]. and at focal adhesions in response to Ras activation. Moreover, In addition to the inside out signalling effects of Ras on inte- PREL1 directly binds to activated Ras in a phosphoinositide- grin activation, engagement of integrins, e.g., through binding dependent manner. Thus, our data pinpoint PREL1 as the first to fibronectin, leads to Ras- and subsequent Erk activation, direct link between Ras signalling and cytoskeletal remodelling which coincides with transient association of Ras with the sites via Ena/VASP proteins during cell migration and spreading. of integrin engagement [2,18]. Moreover, Nobes and Hall 2004 Federation of European Biochemical Societies. Published showed that Ras- but not Erk activation is required for the on- by Elsevier B.V. All rights reserved. set of cell migration in assays, as exemplified by microinjection of neutralizing anti-Ras antibodies and chemi- Keywords: Ras; VASP; Mena; Actin; Lamellipodium; Focal adhesion cal inhibitors of MEK [2]. Nevertheless, the molecular link from Ras to actin reorganization and adhesion turnover dur- ing cell motility is poorly defined. Ena/VASP proteins are important regulators of the actin polymerization machinery, although the exact mechanism of 1. Introduction how this regulation occurs is under debate [12,19]. They are localized to focal adhesions, along stress fibres and at the tips The actin cytoskeleton is a prerequisite for cell motility and of protruding lamellipodia and filopodia [9,20–22] and are undergoes various rearrangements during lamellipodia and fil- binding partners of the actin monomer binding protein profilin opodia formation, bundling into stress fibres or anchorage to [21,23]. Combined genetic inactivation of the Ena/VASP fam- the substrate through focal adhesions. Cell migration is medi- ily members VASP and Mena indicated that these proteins ated by a concerted interplay of assembly and disassembly of may not be essential for the formation of lamellipodia and fil- all these structures, the formation of which is tightly regulated opodia, although fibroblasts lacking these family members dis- by small of the Rho family [1,2]. The protrusion of play alterations in migratory behaviour and protrusion lamellipodia and filopodia requires actin polymerization efficiency [11,12]. Actin-based motility of the facultative intra- involving the de novo nucleation of actin filaments and the cellular pathogen Listeria monocytogenes is enhanced by Ena/ addition of monomers to the barbed ends of pre-existing fila- VASP proteins, both in vivo [24] and in vitro [25]. Ena/VASP ments [3,4]. Incorporation of actin into dynamic structures is proteins interact directly with the Listeria surface protein ActA restricted to the sites, e.g., the lamellipodial tip [5], where the and the enhanced motility was attributed to the recruitment of fast growing (barbed) ends of the filaments are located [6]. profilactin by VASP [23,26] to promote localized actin assem- There is accumulating evidence for the presence of multi pro- bly [27]. However, in vitro, Ena/VASP proteins can also en- tein-complexes at the very edges or tips of protruding lamelli- hance actin based motility in the absence of profilin [19]. The surface protein ActA of Listeria is essential and suffi- *Corresponding author. Fax: +49 531 6181 444. cient for the intra- and inter-cellular actin-based motility of E-mail address: [email protected] (T.E.B. Stradal). this pathogen. A poly-proline motif flanked by hydrophobic

0014-5793/$22.00 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2004.10.110 456 A. Jenzora et al. / FEBS Letters 579 (2005) 455–463 and acidic residues was identified to mediate recruitment of as a template and was obtained from Ambion (Huston, TX). The ex- Ena/VASP proteins by binding to their N-terminal EVH1 do- pressed sequence tag (EST) clone (Acc. No.: 14500382) was obtained mains [28]. Moreover, homologous polyproline repeats in zyx- from the Resource Centre of the German Human Genome Project (RZPD, Berlin, Germany) and contained the full length coding se- in [29] and vinculin [30] were shown to be involved in the quence of the murine gene. targeting of Ena/VASP proteins, via their EVH1 domains, to Murine full length PREL1 derived from the above EST clone was focal adhesions. sub-cloned into pEGFP-C or pEGFP-N vectors (Clontech, Palo Alto, Additional proteins harbouring EVH1-binding motifs were CA). Constructs encoding the proline-rich N-terminus (residues 1–252) and C-terminus (residues 398–668), the RA-domain (residues 151–276) identified more recently, for instance the haematopoietic pro- and the PH-domain (residues 301–433) were generated by PCR and tein Fyb/Slap/ADAP, which binds to Ena/VASP family pro- cloned into pEGFP-C (Clontech) and pGEX-6P (Amersham) vectors teins upon T-cell receptor activation [31], and palladin, also for eucaryotic and bacterial expression, respectively. All sequences implicated in contributing to subcellular Ena/VASP position- were confirmed by sequencing. An overview of the constructs used in ing [32]. However, it remained unclear as to which interaction this study is available in the Supplementary Information. partners Ena/VASP proteins may have at lamellipodia tips and 2.3. Antibodies how they are recruited to this subcellular compartment. Re- The monoclonal antibody (subtype IgM, clone 31C4) recognizing cently, for instance, the proto-cadherin Fat1 was found to be the poly-proline region of zyxin was generated against recombinant capable of interacting with Ena/VASP proteins at these sites, human full length zyxin [34]. Epitope mapping was performed as de- providing a potential mechanism for Ena/VASP recruitment scribed earlier [40]. to lamellipodia [33]. Polyclonal rabbit antiserum termed pcVASP1 was raised against the synthetic peptide: C-ATQVGEKPPKDESASQEESEARLPAQ de- The starting point of the present study was our finding that a rived from the murine VASP sequence. The anti-PREL1 antiserum monoclonal antibody raised against zyxin recognized the re- pcPREL1 was raised against a bacterially expressed GST-tagged frag- peated EVH1-binding consensus (D/E-FPPPP-XD/E) in both ment of PREL1 comprising residues 1–420. Antisera were affinity puri- zyxin and vinculin, and stained lamellipodia tips in migrating fied using the respective peptides or recombinant proteins immobilized fibroblasts, sites lacking both zyxin and vinculin [34]. Using on CNBr-sepharose 4B (Amersham Biosciences, Sweden). Specificity of the antisera was confirmed by Western blot detection of the endog- this antibody, we identified a novel protein from a HeLa enous and ectopically expressed GFP-tagged proteins. Monoclonal expression library with high similarity to the C. elegans anti-Mena antibody was described earlier [21]. Antibodies specific MIG-10 protein [35,36], which we have tentatively termed for GFP and Zyxin [34] were from Synaptic Systems (Go¨ttingen, Ger- PREL 1 for Proline-Rich EVH1 Ligand 1. many). Monoclonal anti-GST antibody was raised using recombinant GST as an antigen and characterized as described [40]. Monoclonal MIG-10 is a 73 kDa protein and shares sequence homology anti-vinculin antibody was from Sigma (Munich, Germany), the with the Grb7-family of signalling adaptors [36–38]. Unlike the monoclonal anti-Ras antibody was from Upstate Biotechnology GRB7/10/14 sub-family, MIG-10 and the novel protein identi- (Charlottsville, VA), and the anti-myc antibody was purchased from fied in this study lack the C-terminal src homology 2 (SH2) do- Abcam (Cambridge, UK). main and harbour multiple poly-proline stretches. We found that PREL 1 not only binds to VASP and Mena and co-local- 2.4. Immunofluorescence, video microscopy, and data processing izes with these proteins at lamellipodia tips and in focal adhe- Cells were plated on glass coverslips coated with either 25 lg/ml lam- inin (Sigma) or 50 lg/ml fibronectin (Roche) prior to either fixation or sions, but that it also interacts with GTP-loaded Ras in a lipid video microscopy as indicated. Immunofluorescence staining was per- dependent manner. These findings thus provide a direct link formed essentially as described [41]. Secondary reagents were Alexa between Ras signalling and remodelling of the actin cytoskele- Fluor488- or Alexa Fluor594-coupled goat antibodies, which were ton via Ena/VASP proteins. used in combination with or without Alexa Fluor-dye coupled phalloi- dins (Molecular Probes, Leiden, The Netherlands) to label the actin cytoskeleton. Video microscopy and microinjection of live B16-F1 and NIH3T3 cells expressing GFP-tagged variants of PREL1 were performed as de- 2. Materials and methods scribed earlier [9]. V12Ras was purified as described [42] and injected at 2.5 mg/ml in a mixture with 0.25 mg/ml Texas-Red-labelled dextrane 2.1. Cells and transfections (70 kDa, Molecular Probes) to control for successful injections. TPA All tissue culture reagents were from Gibco BRL and all chemicals (tetradecanoyl phorbol acetate)-treatments were performed at a con- were from Sigma unless mentioned otherwise. B16F1 (ATCC: CRL- centration of 1 lg/ml. 6323) cells were maintained as described earlier [39]. Rat2 cells (ATCC: Data were acquired using a Zeiss Axiovert 135TV microscope CRL-1764) were cultured in DMEM supplemented with 10% FCS equipped with a back-illuminated cooled charge-coupled device cam- (PAA Gold) and 2 mM glutamine. Swiss 3T3 (ATCC: CCL-92) and era (Princeton Research Instruments) driven by IPLab software (Scan- NIH3T3 cells (ATCC: CRL-1658) were cultured in DMEM supple- alytics, Fairfax, VA) and processed using IPLab and Adobe mented with 10% FBS (Sigma), 2 mM glutamine 1.5% non-essential Photoshop 6.0 (Adobe Systems, San Jose, CA) software. Statistical aminoacids and 1% Na-pyruvate. Transfections were carried out with analyses were carried out using Microsoft Excel 2001 and Sigma Plot SuperFect (Quiagen, Germany, for B16-F1) or with FuGENE (Roche, 8 software. Germany, for Rat2 and NIH 3T3), according to the manufacturerÕs protocols. 2.5. Immunoprecipitations and pull-down assays For immunoprecipitations, cells grown in 10 cm diameter dishes 2.2. Identification of PREL1 and cloning of expression constructs were washed with PBS and lysed in 500 ll of ice-cold lysis buffer L1 Screening of a HeLa cell expression library (#69656-3; Novagen (8 mM Tris base, 12 mM HEPES, 50 mM NaCl, 15 mM KCl, 12 Inc., Madison) was carried out as described earlier [31] using the mM MgCl2, 1 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 1% PEG monoclonal antibody 31C4 (isotype IgM), which recognizes the 6000, and Complete Mini, EDTA-free protease inhibitor cocktail EVH1-recognition consensus, leading to the identification of a novel (Roche) supplemented with or without 1% Triton X-100 as indicated) open reading frame (ORF) with high similarity to the murine ORF for 10 min on ice. In the absence of detergents, cells were harvested prp48 (Acc. No.: AF020313) and the nematode mig-10 sequence. with a cell scraper and lysed by three 10 s pulses in an ultrasonic water Reverse transcription (RT)-PCR was carried out to obtain the full bath (RK 102H, Bandelin electronic, Berlin, Germany) with 5 s vor- length sequence information using the Titan Kit (Roche) according texing steps in between. All lysates were cleared by centrifugation for to the manufacturerÕs instructions. Human brain total RNA was used 15 min at 12 000 · g and 4 C. Cleared lysates were incubated with A. Jenzora et al. / FEBS Letters 579 (2005) 455–463 457

5 lg of the indicated antibodies for 1 h and followed by incubation binding partners of the WW domain of FE65 (PRP48 for Pro- with 30 ll protein G-sepharose beads (Amersham Biosciences) under line Rich Protein from clone number 48) [44]. We tentatively agitation for 45 min at 4 C. Beads were then washed twice with lysis termed this novel protein PREL1 (Proline Rich EVH1 buffer. Precipitates were resolved by SDS–PAGE and analysed by immunoblotting. Ligand 1). For pull-down assays, the recombinant GST-tagged proteins or re- Sequence analyses and database searches revealed that the combinant GST alone as control (for constructs, see Supplementary human and the murine sequences of PREL1 comprise a coding Material) were coupled to glutathione-sepharose beads (Pharmacia) sequence of 1998 and 2007 base pairs, respectively, encoding a and washed in buffer B (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM protein with numerous proline-rich stretches in their N- and C- MgCl2, and 0.1 mM DTT). Final concentration of the recombinant protein on beads was 2 mg/ml slurry. 30 ll of beads was incubated with termini and a calculated molecular mass of 73 and 74 kDa, cleared lysates (see above) for 1 h at 4 C, washed twice with lysis-buf- respectively. In addition, the protein displays a Ras association fer and resuspended in SDS-loading buffer. Precipitates were resolved (RA) and a pleckstrin homology (PH) domain, both of which by SDS–PAGE and analysed by immunoblotting. For precipitations are embedded in a stretch of residues that shows homology to of endogenous Mena in Fig. 3(a) and (c), Rat-2 cells were used instead of NIH3T3 due to the high expression levels of this protein as com- and defines the Grb7/10/14 family of signalling adaptors [36] pared to the latter cell line. (Fig. 1(c)). This region of sequence-homology of approxi- mately 300 amino-acid residues was also identified in the C. 2.6. Protein overlay assays on immobilized peptides and lipids elegans protein MIG-10 and was termed the GM-region 0.2 mg/ml purified recombinant EVH1-domains of Mena, VASP and (Grb7-MIG-10 homology region) [36,38]. MIG-10, the puta- Evl fused to GST [28,43] were incubated on peptide-scans comprising tive C. elegans ortholog of PREL1, has been implicated in neu- either the proline-rich N- or C-terminus of murine PREL1 in TBS-T containing 10% FCS as described earlier [28]. Detection was performed ronal cell migration during embryonic development of the in analogy to Western blot membranes using a monoclonal anti GST nematode [35,45]. antibody. For protein-lipid overlays, 0.01 lg/ml of the purified recom- Two homologous genes are present in mammals, PREL1 binant GST-tagged PH-domain of PREL1 was incubated on PIP-strips identified here and the open reading frame KIAA1681 (which (Echelon Research Inc.) and developed as described above. Binding of we tentatively named PREL2) with calculated molecular the GST-tagged EVH1- and PH domains in overlays was quantified by luminometry employing a cooled CCD-camera (Fuji) and analysed weights of 74 and 134 kDa, respectively. PREL1 and PREL2 using AIDA software (Raytest, Germany). Signals from areas of iden- (KIAA1681) show a high degree of sequence homology also tical size were integrated, background subtracted and normalized to outside the GM region especially in the N-terminus, while 100 as a value for the highest binding. the most striking difference between them is the size of the C-terminal proline-rich extension. (For a detailed alignment 2.7. Spreading assay and Ras activation assay see Supplementary Material.) 10E4 NIH3T3 cells were plated on fibronectin (50 lg/ml, Roche) coated glass coverslips (12 mm diameter) in 24 well plates and fixed The C. elegans genome harbours one PREL-like gene, i.e., with 4% PFA/PBS after different time points as indicated. Cells were MIG-10, with significant homology to PREL1, while the only permeabilized with 0.1% TX-100 in 4% PFA for 45 s, washed with PREL-like gene detectable in the genomes of Drosophila mela- PBS and stained with the polyclonal anti PREL1 antibody as described nogaster (CG11940) and Anopheles gambia (agCP1621) display above. At 10, 20, 40 or 60 min after plating, the number of cells that higher homology to PREL2. Thus, these two novel proteins, had already initiated spreading was quantified. In addition, the frac- tion of spreading cells was scored for PREL1 localization at lamellipo- mammalian PREL1 and PREL2, may constitute a new protein dia tips for each timepoint (n P 300). In parallel, 2x10E6 NIH3T3 family together with MIG-10 and the gene products encoded cells were plated on fibronectin coated (20 lg/ml, Roche) 10 cm diam- by the fly database entries. eter dishes and subjected to Ras activation assays after different time Interestingly, PREL2 (KIAA1681) is located on a region of points according to the manufacturerÕs instructions (Upstate Biotech- nology). Levels of GTP-Ras were assessed by luminometry employing human chromosome 10, which has been implicated in the a cooled CCD-camera (Fuji) and analysed using AIDA software (Ray- inherited neuro-degenerative disease Amylotrophic Lateral test, Germany). Sclerosis 2 (ALS2) and was therefore discussed as a potential candidate (ALS2 candidate region 9: ALS2CR9) involved in the development of this disease [46]. 3. Results and discussion

3.1. Identification of PREL1 3.2. Expression pattern, localization and dynamics of PREL1 When characterizing monoclonal antibodies raised against To learn more about the expression pattern, tissue distribu- human recombinant zyxin [34], a clone was identified (see also tion and sub-cellular localization of PREL1, we raised poly- Section 2), which not only recognized zyxin, but also vinculin clonal antibodies against recombinant fragments or synthetic and a number of unidentified bands in Western blots of lysates peptides derived from different regions within the protein. of various cell lines (Fig. 1(a)). In immunofluorescence, this One antiserum characterized in more detail specifically recog- antibody stained focal adhesions and stress fibres of fibroblas- nized endogenous PREL1, ectopically expressed GFP-tagged tic cells, where zyxin and vinculin are enriched, as well as the PREL1 as well as the recombinant protein and revealed an tips of protruding lamellipodia (Fig. 1(b)). apparent molecular weight of PREL1 of 100 kDa on Western In order to identify novel ligands for Ena/VASP proteins, we blots (Fig. 2(a)). Western blot analyses of different cultured cell screened a HeLa expression library employing the above anti- lines and various mouse tissues (Fig. 2(b)) using the novel body. From this screen, we derived several crossreactive polyclonal antiserum showed that PREL1 is widely expressed clones, which were identified as the Rho-GEF LARG (Acc. and enriched in the haematopoietic system. No.: NP_056128), the ERM family member Radixin (Acc. Immunolabelling experiments using affinity purified poly- No.: NP_002897) and a novel protein, with sequence similarity clonal anti-PREL1 antibodies revealed a strong signal at lamel- to the murine ORF prp48 (Acc. No.: AF020313), which was lipodial tips and in addition at focal adhesions in Swiss 3T3 identified earlier (together with Mena) in a screen for potential fibroblasts growing on fibronectin (Fig. 2(c)). To further con- 458 A. Jenzora et al. / FEBS Letters 579 (2005) 455–463

(Fig. 2(d)) reminiscent of the distribution of VASP during lamellipodia protrusion [9] and, less prominently, to focal adhe- sions (Fig. 2(d)). Notably however, while PREL1 robustly tar- geted to the tips of protruding lamellipodia in B16-F1 and Swiss 3T3 cells, video microscopy of GFP-PREL1 in NIH3T3 cells (not shown) or immunolabelling of endogenous PREL1 in the same cells revealed a more complex distribution pattern, since lamellipodia protrusion was not always coincident with PREL1 recruitment to their tips. Therefore, we conclude that PREL1 is targeted to focal adhesions and lamellipodia in a cell type and/ or signalling status-specific manner (see also Figs. 5 and 6).

3.3. Interaction of PREL1 with Ena/VASP proteins via N-terminal and C-terminal poly-proline motifs To test for a direct interaction of PREL1 with Ena/VASP proteins, we performed co-immunoprecipitation experiments using monoclonal anti-Mena and polyclonal anti-PREL1 anti- bodies. In these experiments, the Ena/VASP family protein Mena co-precipitated moderate amounts of ectopically ex- pressed untagged PREL1 and anti-PREL1 antibodies co- precipitated endogenous Mena from the same lysates, suggest- ing that at least a sub-portion of Mena and PREL1 is bound to each other in vivo (Fig. 3(a)). Analogous experiments were performed to analyse the interaction of endogenous PREL1 and the Ena/VASP-family member VASP in NIH 3T3 cells with virtually identical results (not shown). To assess whether the interaction was direct and whether an EVH1 domain/poly-proline interaction accounts for the bind- ing of PREL1 to Mena and VASP as predicted from the sequence of PREL1, we performed pull-down assays with GST-tagged EVH1 domains from lysates of cells expressing dif- ferent GFP-tagged constructs comprising either the N-terminal or C-terminal proline-rich motifs of PREL1. In these pull-down assays, significant amounts of both the N-terminus and the C-terminus of PREL1 as well as the endogenous full length pro- tein or the ectopically expressed protein fused to GFP could be precipitated from cellular lysates (Fig. 3(b)). In addition, we Fig. 1. PREL1 is a novel proline rich protein. (a) Identification of a novel proline rich protein with an apparent molecular weight of 100 performed pull-down assays using the recombinant GST- kDa in lysates of murine NIH3T3 fibroblasts employing a monoclonal tagged proline-rich N- and C-terminus of PREL1. Endogenous antibody (31C4, subtype IgM) that recognizes the EVH1-recognition Mena and VASP were readily coprecipitated from lysates of motifs in zyxin and vinculin. Detection of zyxin (75 kDa) and vinculin Rat-2 and NIH3T3 cells, respectively (Fig. 3(c)). (120 kDa) was confirmed employing specific antibodies, respectively, To further identify those poly-proline motifs of PREL1 that as indicated. Note that endogenous PREL1 is not detectable in lysates of murine B16-F1 melanoma cells. (b) In Swiss 3T3 fibroblasts growing mediate direct binding to the EVH1 domains of Ena/VASP on fibronectin, the 31C4 antibody stained both focal adhesions and the family proteins, we employed the peptide overlay technique very edge of lamellipodia (arrowhead), as confirmed by counterstain- [28] (Fig. 3(e) and (f)). The proline rich sequences of the N-ter- ing of the actin cytoskeleton with phalloidin. This distribution is highly minus (residues 1–160) as well as the C-terminus (residues 433– reminiscent of the localization pattern of Ena/VASP family proteins as exemplified by labelling with polyclonal anti-VASP antibodies 668) of PREL1 were synthesized in an immobilized form on a (pcVASP). Scale bars equal 20 lm. (c) Domain overview of the newly membrane as an array of 15-mer peptides with an overlap of identified PREL1 as compared to the C. elegans MIG-10 protein, the 12 AA with the preceding and the subsequent peptides, respec- open reading frame KIAA1681 (PREL2), an uncharacterized Dro- tively [28,47]. sophila open reading frame (CG11940) as well as the Grb7/10/14 Under these conditions, two of the proline rich stretches, one family. The top line marks the fragment of PREL1 that was isolated from a HeLa expression library in the 31C4 screen. The percentages in the N-terminus (Fig. 3(e)) and one in the C-terminus (Fig. display the degree of sequence identity within the domains of the 3(f)), displayed significant binding to GST-tagged recombinant depicted proteins and P indicates the location of poly-proline stretches. EVH1 domains of Mena (Fig. 3(e) and (f)), VASP and EVL All proteins share a central RA (Ras association) and PH (pleckstrin (not shown). In vitro binding of the recombinant EVH1 do- homology) domain embedded in a larger region of homology termed GM region, for Grb7/10/14 and MIG-10 region of homology. mains to the other proline rich motifs was significantly weaker as compared to the motifs highlighted in Fig. 3(e) and (f). In addition, binding to the N-terminal motif was app. 1.5-fold firm this localization, we fused the murine full length cDNA to lower as compared to the C-terminal motif (for signal quanti- EGFP (either N- or C-terminally) and visualized its dynamics in fication, see Section 2). This latter motif also displays the high- B16-F1 cells moving on laminin. These studies confirmed that est degree of conservation to the EVH1 binding consensus PREL1 can indeed be recruited to the tips of lamellipodia motif D/EFPPPPXD/E [28]. A. Jenzora et al. / FEBS Letters 579 (2005) 455–463 459

Fig. 3. PREL1 binds to the EVH1 domains of Ena/VASP proteins in vivo and in vitro. (a) Co-immunoprecipitations of PREL1 and Mena using either the polyclonal anti PREL1 antibody pcPREL1 or a monoclonal anti Mena antibody [21]. (b) Pull-down experiments with the GST-tagged EVH1 domain of Mena from lysates of NIH3T3 cells expressing the GFP-tagged proline-rich N- (PR-N) and C-termini (PR- C) or GFP-tagged full length PREL1. The EVH1 domain readily precipitated both the N- and C-terminal proline-rich domains of Fig. 2. Expression pattern and subcellular distribution of PREL1. PREL1 as well as the endogenous and the GFP-tagged full length (a,b) Western blotting of PREL1 from extracts of different cultured cell protein. Western blot detection was performed using either a mono- lines (a) and various mouse tissues (b) revealed that PREL1 is widely clonal anti-GFP antibody or polyclonal anti-PREL1 antibodies expressed and highly enriched in tissues or cells of haematopoietic (pcPREL1) as indicated. (c) Pull-down experiments of Ena/VASP origin. (DMBM: bone marrow macrophages, DCS: dendritic cells). proteins using the GST-tagged proline rich N- (PR-N) or C-terminus Tubulin detection was used as loading control. Note that GFP-tagged (PR-C) of PREL1. Lysates from Rat2 cells expressing endogenous and untagged PREL1 are readily expressed in B16F1 cells lacking the Mena and from NIH3T3 cells expressing high levels of endogenous endogenous protein. A Western blot loaded with various samples and VASP were challenged with the immobilized GST-tagged proline rich showing the whole molecular weight range can be viewed in the N- or C-terminus of PREL1. Western blot detection was performed Supplementary Material. (c) Swiss3T3 cells were immunolabelled with using either a monoclonal anti-Mena antibody [21] or polyclonal anti- polyclonal anti-PREL1 antibodies (pcPREL1) and counterstained for VASP antibodies (pcVASP). Endogenous VASP and Mena were filamentous actin with phalloidin as indicated. In these cells, PREL1 is readily co-precipitated. (d) Control pull-down experiments using localized at the very edge of lamellipodia (arrowhead) and in focal immobilized GST alone from lysates of NIH 3T3 cells transfected adhesions (arrow). Scale bar equals 10 lm. (d) Time lapse phase with different GFP-tagged PREL1 constructs. Cell lysates (1) were contrast (right panels) and fluorescence (left) microscopy of B16-F1 loaded next to the GST pulldowns (2) and Western blot detection was mouse melanoma cells transfected with EGFP-tagged PREL1 moving performed using the monoclonal anti GFP antibody. Note that GST on laminin. The panels display representative frames at three consec- does not co-precipitate the GFP-PREL1 or truncated mutants. (e,f) utive timepoints during protrusion of the cell front. Time is given in Peptide overlays of the proline-rich N-terminus (e) or C-terminus (f) of minutes and seconds. Note the recruitment of GFP-tagged PREL1 to PREL1 with the GST-tagged EVH1 domain of Mena. Boxes and the lamellipodium tip (arrowheads) and less prominently to focal arrows depict the amino acid sequences of the spots showing the adhesions (arrow). The bracket in the bottom phase contrast image strongest signals within the N- or C-terminus of PREL1 as indicated. marks the width of the lamellipodium. Scale bar equals 5 lm.

3.4. Interaction of PREL1 with phosphoinositides and phosphates and to be targeted to membranous compartments Ras-GTPases [48]. In order to test for potential phospholipid binding of The central region of PREL1 comprises a RA and a PH do- the PREL1-PH domain, we overlayed a membrane with immo- main embedded within the GM region of homology (see also bilized phospho-lipids (PIP Strip, Echelon Research Inc.) with Fig. 1(c)). A common feature of PH domains, which occur the recombinant GST-tagged PH domain of PREL1, in anal- in a wide range of proteins, is their ability to bind inositol ogy to the peptide overlay technique. 460 A. Jenzora et al. / FEBS Letters 579 (2005) 455–463

Excitingly, the PH domain of PREL1 displayed highest (Fig. 4(c), lower panel). Members of the Rho family did not binding affinity to phosphatidylinositol monophosphates co-precipitate with PREL1 (not shown), and even the Ras- PtdIns(5)P and PtdIns(3)P (Fig. 4(a)), which reflects the bind- family GTPase Rap1 was not detectable in immunoprecipi- ing characteristics shown for the same domain in Grb7 [49]. tates of PREL1 under these conditions (Fig. 4(c)), indicating Binding of the GST-tagged PH domain to immobilized lipids that binding of PREL1 to small GTPases may be highly spe- was quantified luminometrically and revealed signals for cific also within the Ras-subfamily. Together, these findings PtdIns(5)P > PtdIns(3)P > PtdIns of 100, 65.8 and 47.6 (arbi- indicate that binding of PREL1 to Triton-soluble components, trary units), respectively. such as most phospholipids, is required for efficient binding of We then tested whether the RA domain of PREL1 might the RA domain to activated Ras-GTPases. bind to Ras-family GTPases. RA domains have been identified From these data, we hypothesize that membrane binding of in RasGTP effector proteins such as RalGDS [50,51]. Some the PH domain might cooperate with the RA domain to asso- downstream effectors of Ras GTPases, such as Nore1/RapL, ciate with RasGTP during signal transmission. Moreover, a have the ability to bind to GTP loaded H-, K- and N-Ras as potential dependency of RA domain binding to RasGTP on well as to Rap1 via their RA domain and to act in a cell type the presence of an intact PH domain and phosphoinositides specific manner [52,53]. Interestingly, the highly similar RA might well explain results from an earlier study, in which domains of Grb7/10/14 proteins were not found to bind to RA-domains of the Grb-family were reported not to bind RasGTP [54,55]. Very recently, however, full length Grb7 as Ras GTP-ases [37,54]. This view is not only supported by the well as a N-terminal fragment of the human ortholog of high sequence conservation of the RA-PH domain tandem be- PREL1, which includes the RA but not the PH domain, was tween PREL1/2 and the Grb7/10/14 proteins, but also by the reported to bind to Ras in pull-down assays [56]. To test for striking similarity in PtdIns phosphate binding characteristics the Ras-binding capabilities of the RA domain of PREL1, of the isolated PH domains of both sub-families. In the light we first performed pull-down assays from lysates of cells of these findings, it will be interesting to re-evaluate the expressing constitutively active mutants of various Ras super- GTPase binding characteristics of all Grb7/10/14 family mem- family GTPases using the recombinant GST-tagged RA do- bers. Finally, in an independent study, Lafuente and colleagues main of PREL1. Under these conditions, GST-RA of have now described the human ortholog of murine PREL1 as PREL1 co-precipitated only low amounts of H-RasV12 (Fig. an interactor of Rap1 in Jurkat T-cells and termed this protein 4(b)) and K-RasV12 (not shown). RIAM (for Rap interacting adaptor molecule) [57]. In yeast To test whether PREL1 can associate with members of the two-hybrid assays, these authors also detected an interaction Ras family of GTPases in vivo, we performed co-immunopre- with H-Ras, albeit weaker than that with Rap1. Resolving cipitations of endogenous PREL1 with ectopically expressed the discrepancies between the observations from the latter myc-tagged constitutively activated mutants of Ras, Rap1, and the present study requires further investigations. Rac1, Cdc42 or RhoA using the polyclonal anti-PREL1 anti- serum. Notably, full length PREL1 could co-immunoprecipi- 3.5. Localization of PREL1 coincides with Ras activation in tate significant amounts of RasV12 (Fig. 4(c)), but only in NIH3T3 cells the complete absence of detergents, while even low amounts Due to the ability of PREL1 to bind to RasGTP, we asked of Triton-X100 or NP-40 completely abolished this interaction as to whether its localization or recruitment might be regulated

Fig. 4. PREL1 binds to constitutively active Ras in a lipid-dependent manner. (a) A PIP-strip was overlayed with the PH domain of PREL1 fused to GST. Detection of bound protein was performed using a monoclonal anti-GST antibody followed by peroxidase-coupled secondary reagents and chemiluminescence. The signal was detected and quantified using a CCD camera (see text for further details). Strong binding was detected for phosphatidyl monophosphates (PtdIns(3)P and PtdIns(5)P) and, albeit weaker, for phosphatidyl inositide (PtdIns). (b) Pull-down experiments using the recombinant GST-tagged RA domain of PREL1 performed on lysates of NIH3T3 cells expressing myc-tagged RasV12, lysed in the presence and absence of Triton X-100 (TX-100). (c) Co-immunoprecipitations of PREL1 and Ras-family GTPases from lysates of NIH3T3 cells expressing constitutively active versions of H-Ras, K-Ras and Rap1. Immunoprecipitations using the polyclonal anti PREL1 antibody pcPREL1 revealed a specific binding of PREL1 to Ras, but not Rap1 (upper panel). Binding of Ras GTPases to endogenous full length PREL1 was completely abolished in the presence of Triton-X 100 (lower panel). Lanes corresponding to cell lysates (relative loading 1/10) and immunoprecipitates are labelled with 1 and 2, respectively. A. Jenzora et al. / FEBS Letters 579 (2005) 455–463 461 by cellular RasGTP levels. This possibility seemed attractive, since it might explain for the differences in subcellular recruit- ment under different conditions and in different cell types (also compare to Fig. 2). It was previously shown that spreading of NIH3T3 cells on fibronectin coincides with increased Ras activity, reaching a maximum in the first 10 min and then declining to basal lev- els within 60 min [58]. Under these conditions, Ras activation was also reported to be accompanied by Erk (extracellular signal regulated kinase) activation [58]. Moreover, Nobes and Hall reported that Ras- and Erk activation during cell migration, induced by wounding of cell monolayers, is re- quired for efficient translocation of the cells into the wound [2]. To learn more about the recruitment mechanisms of PREL1, we studied the subcellular distribution of PREL1 in NIH3T3 cells during spreading on fibronectin in the context of cellular RasGTP levels, as determined by Ras activation assays. The time course of Ras activation in NIH3T3 cells is shown in Fig. 5(a) and is virtually identical to the results obtained in ear- lier studies [58]. Intriguingly, microscopic analysis of cells, pro- cessed in parallel in order to study the subcellular distribution of endogenous PREL1, revealed a clear recruitment of PREL1 to the tips of circular lamellipodia typically occurring during cell spreading on fibronectin (Fig. 5(b)). To assess if the num- ber of cells displaying lamellipodial recruitment of PREL1 cor- relates with the levels of cellular Ras-activation, we performed detailed quantifications of the percentage of cells that are spread at a given timepoint (line plot in Fig. 5(c)) as compared to the percentage of spread cells that display lamellipodial tip localization of PREL1 (column plot in Fig. 5(c)). A clear cor- relation of high Ras activity and PREL1 localization during spreading could be observed. After 60 min, when cellular Ras- GTP levels had returned to a basal level, cells displaying lam- ellipodial PREL1 localization were observed only rarely, while Ena/VASP proteins still displayed the typical subcellular distri- Fig. 5. Actin cytoskeletal targeting of PREL1 correlates with Ras bution (lower left image in Fig. 5(b)) described in earlier stud- activation during cell spreading. (a) Ras activity in spreading NIH 3T3 ies [9,20]. Hence, we conclude that PREL1 is not essential for fibroblasts was quantified at different timepoints after plating on fibronectin as indicated, employing a Ras activation assay based on the constitutive recruitment of Ena/VASP proteins to lamelli- Ras-GTP pulldowns using the GST-tagged Ras binding domain podia or focal adhesions, but instead is recruited to the sites (RBD) of Raf1. (b) Localization of PREL1 during spreading on of rapid actin turnover during Ras activation, where it may fibronectin was determined by indirect immunofluorescence at different then modulate actin assembly by interaction with Ena/VASP timepoints after plating on fibronectin-coated coverslips using poly- proteins. clonal anti PREL1 antibodies (pcPREL1). Note that the recruitment of PREL1 to lamellipodia is largely lost at late stages of spreading To further corroborate the coincidence between Ras activa- (upper right image), while VASP remained prominently associated tion and the translocation of PREL1 to sites of cellular actin with lamellipodia and focal adhesions (lower right image). Scale bars assembly, such as lamellipodia and focal adhesions, we exper- equal 10 lm. (c) Quantification of the percentage of spread cells at the imentally increased Ras activity in these cells by either micro- timepoints indicated (line plot) as compared to the percentage of spread cells displaying lamellipodial tip localization (column plot). injection of constitutively active recombinant RasV12 (Fig. Values are means ± standard errors of means from three independent 6(a)) or by TPA treatment (Fig. 6(b)), which was described ear- experiments (n P 300 for each condition). lier to significantly increase Ras-GTP levels in NIH3T3 cells [59]. In response to both treatments, PREL1 was clearly re- cruited to focal adhesions, confirming that PREL1 indeed levels. We map in detail the binding sites of the EVH1 do- associates with the actin cytoskeleton in response to Ras acti- mains of Ena/VASP proteins on the proline rich N- and C- vation. termini of PREL1, in addition to characterizing the binding features of its central RA - and PH domain. We show that PREL1 specifically associates with Ras via its RA domain 4. Conclusions in a lipid dependent fashion, the latter of which is most likely attributable to the adjacent PH domain. Finally, we demon- We here report the identification and characterization of a strate that the subcellular targeting of PREL1 to lamellipodia novel proline rich protein, capable of interacting with the and focal adhesions correlates with high Ras-GTP levels as Ena/VASP family of proteins and of targeting to lamellipo- induced for instance during cell spreading or by phorbol es- dia tips and focal adhesions coincident with high RasGTP ter treatment. 462 A. Jenzora et al. / FEBS Letters 579 (2005) 455–463

Fig. 6. Translocation of PREL1 upon experimentally induced Ras activation. (a,b) Time lapse fluorescence and phase contrast microscopy of NIH 3T3 fibroblasts transfected with GFP-tagged full length PREL1 before (À2 min) and after (+61 min) microinjection of constitutively active Ras V12 (a) or before (À2 min) and after (+24 min) treatment with the phorbol ester TPA (b). Significant translocation of largely cytosolic GFP-PREL1 to focal adhesions upon experimentally induced increase in cellular Ras-GTP levels could be observed. (c) NIH 3T3 cells expressing GFP-tagged PREL1 (green in merge) and counterstained for endogenous VASP (red in merge) as indicated following TPA treatment (15 min). Note the colocalization of PREL1 and VASP under these conditions. Scale bars equal 10 lm.

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